Antigen protein HPB of bovine-derived B-type pasteurella multocida and preparation method and application of recombinant protein of antigen protein HPB

Abstract

The invention provides an antigen protein HPB of bovine-derived B-type pasteurella multocida and a preparation method and application of recombinant protein of the antigen protein HPB. The amino acid sequence of the antigen protein HPB is shown as SEQ ID NO.3. The preparation method of the antigen protein HPB comprises the following steps: 1) cloning a coding gene sequence, shown as SEQ ID NO.1, of the antigen protein HPB after codon optimization into a prokaryotic expression vector; a recombinant plasmid containing the antigen protein HPB coding gene is obtained; transforming the recombinant plasmid into escherichia coli competent cells to obtain a recombinant expression strain; dyeing the strain into expression cells; the antigen protein HPB is obtained through fermentation culture, inducible expression and purification. Through a challenge protection test on mice, the protection rate of the mice can reach 100%, and the problem of poor protection effect of the B-type pasteurella multocida subunit vaccine for bovine hemorrhagic septicemia at present is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of veterinary biological products and relates to a preparation method and application of a bovine-derived antigen protein HPB of type B Pasteurella multocida and a recombinant protein HPB. Background Art

[0002] Pasteurella multocida (Pm), a member of the genus Pasteurella in the family Pasteurellaceae, is a significant Gram-negative bacterium that infects a wide range of livestock, poultry, wildlife, and humans, causing significant morbidity and mortality. This pathogen has numerous serotypes, classified as A, B, D, E, and F based on differences in capsular antigens. Depending on the serotype and host species, it can manifest as bovine hemorrhagic septicemia, bovine pneumonia, swine pleuropneumonia, swine atrophic rhinitis, fowl cholera, rabbit hemorrhagic septicemia, and meningitis. In Chinese cattle, hemorrhagic septicemia is primarily caused by type B Pasteurella multocida, which occurs sporadically and occasionally in an endemic manner. This disease, characterized by lung lesions, is an acute infectious disease that severely impacts the cattle industry. It is classified as a Category II animal disease in my country, with a mortality rate of up to 80% to 90%. This disease causes significant economic losses to the cattle industry.

[0003] Currently, my country only has an inactivated Pasteurella multocida type B vaccine for preventing bovine hemorrhagic septicemia. However, inactivated vaccines have poor safety and are prone to the risk of virus shedding. Therefore, there is an urgent need to develop a subunit vaccine for Pasteurella multocida type B. However, there are currently few reports on the development of subunit vaccines for Pasteurella multocida type B in China and abroad. Summary of the Invention

[0004] We used reverse genetics to analyze the surface proteins of Pasteurella multocida and discovered a relatively conserved surface protein of type B Pasteurella through screening. The recombinant protein HPB expressed by it was used as an antigen. By immunizing mice, the protection rate reached 100%, solving the problem of poor protection effect of the current type B Pasteurella multocida subunit vaccine for bovine hemorrhagic septicemia.

[0005] According to one aspect of the present invention, the present invention provides an antigen protein HPB of bovine type B Pasteurella multocida, characterized in that the amino acid sequence of the antigen protein HPB is shown in SEQ ID NO.3.

[0006] In a preferred technical solution of the present invention, preferably, the nucleotide sequence of the antigen protein HPB is as shown in SEQ ID NO.1.

[0007] In a preferred technical solution of the present invention, preferably, the antigen protein HPB is a polypeptide encoded by the JDU55_RS05735 gene, which is a conserved hypothetical protein located on the surface of the bacteria.

[0008] According to another aspect of the present invention, the present invention provides a method for preparing the antigen protein HPB, the preparation method comprising the following steps:

[0009] 1) Cloning the codon-optimized HPB coding gene sequence as shown in SEQ ID NO. 1 into a prokaryotic expression vector to obtain a recombinant plasmid containing the HPB coding gene;

[0010] 2) transforming the recombinant plasmid into competent E. coli cells to obtain a recombinant expression strain; and then transfecting the recombinant expression strain into expression cells; and

[0011] 3) After fermentation, expression induction, and purification, the antigen protein HPB shown in SEQ ID NO. 4 was obtained.

[0012] In a preferred technical solution of the present invention, preferably, in step 1), the prokaryotic expression vector is pET30.

[0013] In a preferred technical solution of the present invention, preferably, in step 2), the Escherichia coli is BL21 (DE3).

[0014] According to another aspect of the present invention, the present invention provides a use of the antigen protein HPB in preparing a Pasteurella multocida type B vaccine for diagnosing, preventing and treating bovine hemorrhagic septicemia.

[0015] The present invention has the following beneficial effects: 1) The conserved polypeptide on the bacterial surface screened by the present invention has a high protection rate as an antigen, solving the problem of poor protection of the current Pasteurella type B subunit vaccine for bovine pneumonia. 2) The antigen protein of the present invention is easy to prepare and can be expressed in a soluble form with high yield. 3) The antigen protein of the present invention is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Alignment of HPB nucleotide sequences before and after optimization.

[0017] Figure 2 pET30-OPTI-HPB double enzyme digestion results: plasmids 1-2 were double digested with XbaI / EcoRI, with band sizes of 5230 bp and 1062 bp, respectively;

[0018] Figure 3The SDS-PAGE test results of the purified HPB protein are shown in Figure 1: 1 is the purified HPB protein, 2 is the marker;

[0019] Figure 4 The Western-blot results of the purified HPB protein are shown in Figure 1: Marker, and 2: HPB protein. DETAILED DESCRIPTION

[0020] The present invention will be further described below with reference to the accompanying drawings and embodiments. The embodiments of the present invention are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention.

[0021] The strains, plasmids and reagents used in the examples of the present invention are all commercially available products.

[0022] Example 1: Clone Construction

[0023] 1.1. Selection of HPB protein

[0024] The Pasteurella antigen protein HPB is a polypeptide encoded by the JDU55_RS05735 gene. It is a conserved hypothetical protein located on the surface of the bacteria. There are currently no reports on this protein.

[0025] We infer that this protein may be a good candidate vaccine antigen. Currently, there is no report on the soluble expression and purification of this protein in a prokaryotic expression system, and the immunogenicity of the protein has not been verified, which is also an important technical problem to be solved by the present invention.

[0026] 1.2. HPB protein codon optimization

[0027] In this laboratory, the complete gene sequence of Pasteurella type B strain PM-1 was used as a reference (GenBank: NZ_CP066223.1). The nucleotide sequence encoding the hypothetical protein of Pasteurella lipoprotein JDU55_RS05735, as shown in SEQ ID NO. 2, was codon-optimized and synthesized. To facilitate purification, a 6×His sequence was added to the C-terminus to obtain the OPTI-HPB sequence, as shown in SEQ ID NO. 1. The nucleotide sequence before and after optimization had a 24.5% nucleotide difference. Figure 1 As shown, the sequence synthesis work was commissioned to Nanjing GenScript Biotechnology Co., Ltd.

[0028] The expression level of nucleotide sequences that have not been codon-optimized is very low.

[0029] 1.3. Construction of pET30-OPTI-HPB recombinant plasmid

[0030] 1.3.1. PCR amplification of target fragment OPTI-HPB

[0031] 1.3.1.1 PCR reaction

[0032] (1) Primer design and synthesis

[0033] Upstream primer: 5'-ATA CATATG TCATTACTAATAACAGCTTGTAG-3'

[0034] Downstream primer: 5'-CTC GAATTC TTAGTGGTGATGGTGG-3'

[0035] (2) The sample loading system is 50 μL, as shown in Table 1 below:

[0036] Table 1

[0037]

[0038] PCR amplification procedure:

[0039]

[0040]

[0041] 1.3.1.2. Gel recovery of PCR products

[0042] (1) Label the sample collection EP tube, adsorption column, and collection tube;

[0043] (2) Weigh the marked empty EP tube and record the value;

[0044] (3) Carefully cut the single target DNA band from the agarose gel using a scalpel and place it into a clean 1.5 mL centrifuge tube;

[0045] (4) Add 600 μL of PC buffer to the 1.5 mL centrifuge tube in step (3) and place in a 50°C water bath for about 5 min, gently turning the centrifuge tube upside down to ensure that the gel is fully dissolved;

[0046] (5) Column equilibration: Add 500 μL of equilibration solution BL to the adsorption column CB2 (the adsorption column is pre-placed in the collection tube), centrifuge at 12,000 rpm / min for 1 min, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube;

[0047] (6) Add the solution obtained in step (5) to the adsorption column CB2, let it stand for 2 minutes, centrifuge at 10,000 rpm / min for 30 seconds, discard the waste liquid in the collection tube, and then place the adsorption column CB2 into the collection tube;

[0048] (7) Add 600 μL of PW buffer to the adsorption column, let it stand for 3 min, centrifuge at 10,000 rpm / min for 30 s, discard the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube;

[0049] (8) Repeat step (7);

[0050] (9) Centrifuge the empty adsorption column at 12,000 rpm / min for 2 min to remove as much rinse solution as possible. Place the adsorption column at room temperature for 10 min to dry thoroughly.

[0051] (10) Place the adsorption column CB2 in the collection tube, add 50 μL of Elution Buffer (preheated at 65°C) dropwise to the middle of the adsorption membrane, let it stand for 3 min, and centrifuge at 12,000 rpm / min for 2 min;

[0052] (11) Remove the centrifuge tube from step (10) from the centrifuge, discard the adsorption column CB2 in the middle, cover the centrifuge tube, and retain the DNA sample in the centrifuge tube;

[0053] (12) Store the DNA sample in step 11 at 4°C and prepare agarose gel electrophoresis to identify the DNA fragments recovered from the gel.

[0054] 1.3.2. Double enzyme digestion reaction of PCR products and vectors

[0055] (1) Label the 1.5 mL EP tubes to be used, add the sample and mix thoroughly according to Table 2 below: 50 μL reaction system.

[0056] Table 2

[0057]

[0058]

[0059] (2) Place the 1.5 mL EP tube in step (1) in a constant temperature water bath at the optimal temperature for the corresponding enzyme and keep it in the water bath for 2-3 hours.

[0060] Gel recovery of double enzyme digestion products: Take out the double enzyme digestion system and perform agarose gel electrophoresis to recover the DNA fragments therein. The method is the same as the gel recovery of PCR products in 1.3.1.2.

[0061] 1.3.3 Ligation reaction

[0062] (1) Prepare several clean 1.5 mL EP tubes, label them, and place them in an EP tube rack for use.

[0063] (2) Add the sample to a 1.5 mL EP tube according to Table 3 below and mix thoroughly.

[0064] Table 3

[0065]

[0066] (3) After adding the sample according to the table in step (2), place each 10 μl reaction system in a 16°C low-temperature cooling liquid circulation machine and water bath for 10-16 hours;

[0067] (4) Take out the EP tube from step (3) and place it in a 65°C water bath for 15 minutes;

[0068] (5) Take out the EP tube from step (4) and store it at 4°C.

[0069] 1.3.4 Conversion reaction

[0070] (1) Quickly add 10 μL of ligation reaction solution to 100 μL of competent cells, pipette to mix, and incubate on ice for 30 min;

[0071] (2) Remove the sample tube and place it in a 42°C water bath for 100 seconds, then immediately place it in an ice bath for 2 minutes;

[0072] (3) Remove the sample tube and add 600 μL of liquid LB medium to the sample tube in a clean bench. Then place the sample tube in a 37°C constant temperature shaker at 220 rpm / min for 1 h.

[0073] (4) Plate coating: Take out the sample tube in step (3), centrifuge at 8,000 rpm / min for 2 min at room temperature, remove 600 μL of supernatant, and resuspend the bacteria at the bottom of the tube with the remaining supernatant. Place the resuspended bacterial solution in the center of the corresponding transformation plate and spread the bacterial solution in the center of the transformation plate evenly with a bacterial spreader.

[0074] (5) Place the transformation plate prepared in step (4) upright in a biochemical constant temperature incubator and incubate at 37°C for 1 hour. Then, invert the transformation plate and incubate for 15 hours.

[0075] (6) Observe the transformation results.

[0076] 1.3.5 Plasmid extraction and double enzyme digestion identification

[0077] 1.3.5.1 Plasmid Extraction

[0078] (1) Use a 10 μL pipette tip to pick a single colony from the transformation plate and transfer it to 5 mL of LB liquid medium containing ampicillin resistance. Incubate the culture at 37°C, 220 rpm / min, and shake overnight.

[0079] (2) Transfer the bacterial suspension to a 1.5 mL EP tube and centrifuge at room temperature, 12,000 rpm / min, for 2 min. Discard the supernatant.

[0080] (3) Add 250 μL of plasmid extraction reagent P1 buffer to the EP tube in step (2) to thoroughly suspend the bacteria;

[0081] (4) Add 250 μL of P2 buffer to the solution in step (3), immediately gently invert the centrifuge tube 5-10 times to mix, and let it stand at room temperature for 2-4 minutes;

[0082] (5) Add 350 μL of P3 buffer to the solution in step (4) and immediately mix by gently inverting the tube 5-10 times; let it stand at room temperature for 2-4 minutes;

[0083] (6) Centrifuge the solution from step (5) at room temperature, 14,000 rpm / min, for 10 min;

[0084] (7) Transfer the supernatant solution from step (6) to the center of the adsorption column and centrifuge at room temperature, 12,000 rpm / min, for 30 s, and discard the liquid in the collection tube;

[0085] (8) Add 500 μL of Buffer DW1 to the center of the adsorption column and centrifuge at room temperature, 12,000 rpm / min, for 30 s. Discard the liquid in the collection tube.

[0086] (9) Add 500 μL of wash solution to the center of the adsorption column and centrifuge at room temperature, 12,000 rpm / min, for 30 s. Discard the liquid in the collection tube and repeat once.

[0087] (10) Empty adsorption column, centrifuge at room temperature, 12,000 rpm, 2 min.

[0088] (11) Place the adsorption column in a clean 1.5 mL centrifuge tube. Add 30 μL of Elution Buffer to the center of the adsorption membrane. Let stand at room temperature for 5 min. Centrifuge at room temperature at 12,000 rpm for 2 min. Save the DNA solution in the tube.

[0089] 1.3.5.2 Double enzyme digestion identification

[0090] (1) Label the 1.5 mL EP tubes to be used and add the sample according to Table 4 below: 20 μL reaction system.

[0091] Table 4

[0092]

[0093] (2) Place the 20 μL reaction system in the EP tube in step (1) in a 37°C constant temperature water bath for 2 hours.

[0094] (3) The double enzyme digestion system sample in step (2) was subjected to agarose gel electrophoresis to check whether the inserted fragment size was correct; the experimental results are shown in Figure 2 : Plasmid 1-2 was double-digested with XbaI / EcoRI, and the band sizes were 5230bp and 1062bp, respectively, indicating that the construction was correct.

[0095] (4) Select the clone with the correct insert and send it to a sequencing company for sequencing. The plasmid with the correct sequencing result will be saved for future use.

[0096] Example 2: Protein expression

[0097] 1. Transformation: Add 1 μl of the constructed plasmid to 100 μl of competent E. coli BL21 (DE3). Incubate on ice for 30 minutes, then heat shock at 42°C for 90 seconds, followed by another 2-minute ice bath. Shake at 200 rpm at 37°C for 1 hour. Pipette 100 μl of the bacterial solution onto a kanamycin-resistant plate and incubate overnight at 37°C. Induce the plate with a small amount of culture.

[0098] 2. Inducible expression

[0099] 2.1. Pick a single colony and transfer it to 3 ml of LB medium containing kanamycin resistance and culture it at 37°C overnight.

[0100] 2.2. Transfer the cells to 15 ml of kanamycin-resistant culture medium at a ratio of 1:100 and culture at 37°C, 200 rpm for 3.5-4 hours.

[0101] 2.3. Fermentation: Inoculate into 2L of kanamycin-resistant culture medium at a ratio of 1:150 and culture at 37°C, 200 rpm for 3.5-4 hours until OD 600 The value is 0.8-1.0.

[0102] 2.4. Induction: Add IPTG to a final concentration of 0.5 mmol / L and culture at 20°C and 220 rpm for 4 h.

[0103] 2.5. Bacteria Collection Collect the bacterial solution, centrifuge at 8000 rpm for 10 min, and collect the bacteria.

[0104] Example 3: Protein purification

[0105] Lysis buffer: 50 mmol / L NaH2PO4·2H2O, 500 mmol / L NaCl, 5 mmol / L imidazole, 0.05% Tween-20. Dissolve completely in ultrapure water, adjust the pH to 8.0±0.01, filter through a 0.8 μm membrane, and store at 2-8°C.

[0106] Buffer A: 50 mmol / L NaH2PO4·2H2O, 500 mmol / L NaCl, 0.05% Tween-20. Dissolve completely in ultrapure water, adjust pH to 8.0±0.01, filter through a 0.8 μm membrane, and store at 2-8°C.

[0107] Buffer B: 50 mmol / L NaH2PO4·2H2O, 500 mmol / L NaCl, 20 mmol / L imidazole, 0.05% Tween-20. Dissolve completely in ultrapure water, adjust the pH to 8.0±0.01, filter through a 0.8 μm membrane, and store at 2-8°C.

[0108] Buffer C: 50 mmol / L NaH2PO4·2H2O, 500 mmol / L NaCl, 50 mmol / L imidazole, 0.05% Tween-20. Dissolve completely in ultrapure water, adjust the pH to 8.0±0.01, filter through a 0.8 μm membrane, and store at 2-8°C.

[0109] Buffer D: 50 mmol / L NaH2PO4·2H2O, 500 mmol / L NaCl, 400 mmol / L imidazole, 0.05% Tween-20. Dissolve completely in ultrapure water, adjust the pH to 8.0±0.01, filter through a 0.8 μm membrane, and store at 2-8°C.

[0110] 1. Bacterial cell disruption: Add the harvested bacterial cells from fermentation into lysis buffer according to the ratio (1 g bacterial cells: 10 ml lysis buffer), resuspend and mix, then disrupt with a homogenizer and collect the supernatant by centrifugation as the sample.

[0111] 2. Nickel column purification

[0112] 2.1. Nickel Column Equilibration: Take nickel affinity packing and load it into an empty chromatography column. Equilibrate with ultrapure water for 2-3 column volumes (CV), drain the concentrated ethanol storage solution, and then equilibrate with lysis buffer for 2-3 CV.

[0113] 2.2. Sample loading: Load the supernatant after lysis and collect the flow-through.

[0114] 2.3. Equilibrate and rinse the column with 20CV of Buffer A.

[0115] 2.4. Wash impurities: Elute impurities with Buffer B and collect the flow-through until no blue color appears in the Coomassie Brilliant Blue G250 test.

[0116] 2.5. Elution: Elute the target protein with Buffer C and collect the flow-through until no blue color appears when tested with Coomassie Brilliant Blue G250.

[0117] 2.6. Column Cleaning: Elute the target protein with Buffer D and collect the flow-through until no blue color appears in the Coomassie Brilliant Blue G250 assay. Then, wash with water for 5CV and 0.5M NaOH for 2CV. Rinse with ultrapure water until neutral and store in 20% ethanol.

[0118] 2.7. Collect the Buffer C eluate and exchange the target protein into Buffer A by dialysis or tangential flow.

[0119] 2.8 Sterile Filtration: Sterilize the protein after the replacement solution by filtering it through a 0.22μm low protein adsorption membrane. Store at 2-8°C until use.

[0120] 2.9. Protein Concentration and Purity Determination: Protein concentration was determined using the BCA assay. The protein yield was then calculated based on the volume of supernatant used during purification and the total amount of protein obtained after purification. For example, in this example, a fermentation volume of 1000 ml of cells was used, and the volume of purified protein was 120 ml at a concentration of 1 mg / mL, resulting in a calculated protein yield of approximately 120 mg / L. Purity was determined using SDS-PAGE, and the purity was consistently above 90%. This is suitable for large-scale production.

[0121] 2.10 Identification of HPB Protein

[0122] 2.10.1 SDS-PAGE detection

[0123] The protein purified in Example 3 was subjected to SDS-PAGE detection. The HPB protein concentration in the sample used was 2 μg / well. The results are as follows: Figure 3 Shown: From Figure 3 It can be calculated that the SDS-PAGE purity of the purified HPB protein is 95% and the molecular weight is about 38 kDa.

[0124] 2.10.2 Western-blot detection

[0125] The protein purified in Example 3 was subjected to Western Blot detection. The transfer time was 1 hour. The primary antibody used was the 7-day serum No. 574 of the two-inactivated vaccine, the dilution ratio was 1:500, and the incubation time was 1 hour. The secondary antibody used was rabbit anti-bovine, the dilution ratio was 1:5000, and the incubation time was 1 hour. The results are as follows: Figure 4Shown: From Figure 4 The results show that the purified HPB protein can effectively bind to the antibodies in the serum.

[0126] 2.11 HPB protein stability verification

[0127] The purified HPB protein of Example 2.9 (1 mg / ml) was divided into 20 portions, each 0.5 ml. Ten portions were placed in a 4°C refrigerator, and one portion was sampled weekly for 10 consecutive times. Ten portions were placed in a -20°C refrigerator, and one portion was sampled weekly for 10 consecutive times. The protein concentration was determined by BCA after each sampling. The results are shown in Table 5 below:

[0128] Table 5

[0129]

[0130] Judging from the changes in protein concentration, the protein remained basically stable during the two groups of experiments.

[0131] Example 4: Immunogenicity and immune protection effect

[0132] 1. Vaccine preparation

[0133] 1.1. Antigen preparation: The purified recombinant HPB protein was sterilized by filtration through a 0.22 μm filter membrane, and the concentration and purity were tested for later use.

[0134] 1.2. Preparation of aqueous phase: Dilute the HPB protein to an appropriate concentration using 1×PBS according to the HPB protein content in the vaccine and stir for 10 minutes to mix thoroughly.

[0135] 1.3. Oil phase preparation: According to the ratio of oil adjuvant: aqueous phase (v:v) = 54:46, measure an appropriate amount of ISA 201VG adjuvant.

[0136] 1.4. Emulsification: The emulsification temperature is required to be 33±1℃. Turn on the oil phase agitator and set the stirring speed to 350rpm / min. Add the water phase to the oil phase at a uniform speed under stirring conditions and continue stirring for 10 minutes to fully mix the water phase and oil phase to emulsify into a two-way oil emulsion vaccine.

[0137] 1.5. Stabilization: After emulsification, turn off the agitator and place the emulsified vaccine at 20°C to stabilize for 1 hour.

[0138] 1.6. Packaging and storage: Pack according to immunization needs and store at 2-8℃ for future use after passing the inspection.

[0139] 2. Immune challenge experiment

[0140] Ten healthy female BALB / c mice weighing about 16-18 g were randomly divided into three groups, with 5 mice in each group. The vaccine prepared in 1 was used for immunization experiments. A second immunization was performed 21 days after the first immunization. Fourteen days after the second immunization, a 3-fold LD50 dose of Pasteurella multocida type B (ZJHL-PMB) (6×10 7 The mice were challenged with HPB (CFU) and observed for morbidity and mortality within 7 days. All mice in the control group died within 7 days, while all mice in the experimental group survived. The results showed that HPB has excellent immunogenicity, with a 100% protection rate in mice. See Table 6.

[0141] Table 6

[0142] Trial Groups Immunization dose Immunization quantity Challenge dose Number of cases and deaths Experiment 1 50ug / piece 5 <![CDATA[6×10 7 CFU]]> 0 Experiment 2 25ug / piece 5 <![CDATA[6×10 7 CFU]]> 0 control group / 5 <![CDATA[6×10 7 CFU]]> 5

[0143] The present invention is illustrated by the above examples, however, it should be understood that the invention is not limited to the specific embodiments and implementations described herein. These specific embodiments and implementations are included herein to assist those skilled in the art in practicing the present invention. Further improvements and modifications will readily occur to those skilled in the art without departing from the spirit and scope of the present invention, and the present invention is therefore limited only by the content and scope of the appended claims, which are intended to cover all alternatives and equivalents within the spirit and scope of the present invention as defined by the appended claims.

[0144]

[0145]

[0146]

Claims

1. An antigenic protein HPB of bovine type B Pasteurella multocida, characterized in that: The amino acid sequence of the antigen protein HPB is shown in SEQ ID NO.

3.

2. The antigen protein HPB according to claim 1, characterized in that The nucleotide sequence of the antigen protein HPB is shown in SEQ ID NO.

1.

3. The antigen protein HPB according to claim 1 or 2, characterized in that The antigen protein HPB is a polypeptide encoded by the JDU55_RS05735 gene and is a conserved hypothetical protein located on the surface of the bacteria.

4. A method for preparing the antigen protein HPB according to any one of claims 1 to 3, characterized in that: The preparation method comprises the following steps: 1) Cloning the codon-optimized HPB coding gene sequence as shown in SEQ ID NO. 1 into a prokaryotic expression vector to obtain a recombinant plasmid containing the HPB coding gene; 2) transforming the recombinant plasmid into competent E. coli cells to obtain a recombinant expression strain; and then transfecting the recombinant expression strain into expression cells; 3) After fermentation, expression induction, and purification, the antigen protein HPB shown in SEQ ID NO. 4 was obtained.

5. The preparation method according to claim 4, wherein In step 1), the prokaryotic expression vector is pET30.

6. The preparation method according to claim 4, wherein In step 2), the Escherichia coli is BL21 (DE3).

7. Use of any antigen protein HPB according to any one of claims 1 to 3 in the preparation of a Pasteurella multocida type B vaccine for diagnosing, preventing and treating bovine hemorrhagic septicemia.

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